Prevalence of Chlamydia trachomatis Infection among Low- and High-Risk Filipino Women and Performance of Chlamydia Rapid Tests in Resource-Limited Settings

Participants.All FSWs attending the SHC in Iloilo City for routine STI screening for gonorrhea, syphilis, and nongonococcal urethritis (NGU) during six 2- to 3-week periods between August 2002 and March 2006 were invited to join the study. The FSWs worked in nightclubs and massage parlors, and some were also recruited to the study by SHC nurses and doctors in an outreach program. Consecutive patients attending the OB-GYN clinic at WVMC for antenatal care (pregnant women), family planning, or assessment of genitourinary symptoms between October 2005 and March 2006 were also recruited to the study. The SHC and OB-GYN components of the study were both approved by the Cambridge Research Ethics Committee and the Ethics Committee of WVMC. Patients from both clinics were eligible for the study if they were female, at least 18 years of age, willing to participate, literate, and not currently menstruating and had not taken antibiotics within the previous month. Written informed consent was obtained from all participants prior to specimen collection. Participants who withdrew from the study were not recorded.

Specimen collection.For SHC attendees, CS and/or VS specimens were collected from each participant by the attending clinician (Fig. 1). In an outreach program in 2002, the participants collected their own VS specimens. In 2004, subsets of specimens were collected as follows: paired CS specimens only in February 2004 and matched CS (n = 2) and VS (n = 2) specimens in June 2004 for PCR and Clearview Chlamydia MF testing. The VS and CS specimens were tested randomly with respect to order of collection with both PCR and Clearview Chlamydia MF assays. The SHC participants recruited in outreach settings were provided with written and verbal instructions on how to obtain two paired self-collected VS specimens. Between October 2005 and March 2006, only two VS specimens were collected by attending clinicians from each SHC and OB-GYN participant for PCR and/or CRT testing. All specimens were coded to ensure patient anonymity, and test results were conveyed to participants with utmost confidentiality. Specimens for PCR testing were frozen at −20°C at the time of sampling before they were transported to Cambridge and stored at −20°C until analysis. The VS or CS specimens were tested on-site with the rapid tests on the day of swab collection. Personnel performing the PCR and rapid tests were blinded to the order of collection of the two swabs, to comparator test results, and to clinical findings.

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FIG. 1.

Specimen collection and testing algorithm followed in the study. CLVW, Clearview Chlamydia MF (Inverness Medical, Bedford, United Kingdom).

Testing by PCR.CSs and VSs (collected either by the participant [SCVSs] or a clinician [CCVSs]) were collected with the BD Culturette EZ (Becton Dickinson Biosciences, San Jose, CA) and screened for the presence of C. trachomatis DNA with the Amplicor CT/NG PCR-based assay (Roche Molecular Systems, Branchburg, NJ), which targets a conserved region of the cryptic plasmid. All swabs were stored at 4°C within 1 h at the collection site and were transported to the testing site on cold packs. At the testing site, swabs were then placed in 3.0 ml of predispensed M4RT medium tubes of a specimen collection kit (Roche). Specimens were vigorously vortexed and stored overnight at 4°C to enhance the release of the adsorbed cells and tested with the Amplicor assay according to the instructions of the manufacturer. In brief, 100 μl of M4RT medium was added to 100 μl of CT/NG lysis buffer, provided by the manufacturer (specimen preparation kit; Roche) in a 1.7-ml SlickSeal microcentrifuge tube (Bioquote, York, United Kingdom) and mixed well by vortexing for 10 min at room temperature. With a new aerosol barrier tip for each sample, 200 μl of CT/NG specimen diluent was added to each tube, vortexed, and incubated for 10 min at room temperature and kept for less than 2 h before aliquots were transferred to reaction tubes containing the CT/NG reaction mixture (Roche). The reaction volume was 100 μl (50 μl of processed specimens and 50 μl of CT/NG reaction mixture). Samples that were inhibitory to PCR (as shown by failed amplification of the internal control) were diluted 1:1 with sample dilution buffer, heated at 95 to 100°C for 10 min, and then amplified. Amplification was performed with an 9700 thermal cycler (Applied Biosystems, Foster City, CA) according to the following protocol: 1 cycle of 50°C for 2 min and 95°C for 5 min, followed by 36 cycles of 93°C for 20 s, 61°C for 1 min, and 71°C for 40 s and then 1 cycle of 72°C for 5 min, after which samples were held at 72°C (>24 h) until detection.

Testing by Clearview Chlamydia MF.In August 2002 and June 2004, paired CS and VS specimens were tested with the Clearview enzyme immunoassay performed on-site by study personnel according to the instructions of the manufacturer. These results were compared to the PCR results from paired CS specimens.

Testing by CRT.VS specimens from the OB-GYN clinic participants and the FSWs were tested with the CRT by clinic personnel. All testers had passed the proficiency testing requirements according to Clinical and Laboratory Standards Institute guidelines (24). CRT testing was performed as described by Michel et al. (22), with some modifications. Briefly, each swab was subjected to extraction by sequential addition of 400 μl of reagent 1, 300 μl of reagent 2, and 100 μl of reagent 3 to the swab in a tapered sample preparation tube, with gentle mixing between additions. The sample preparation reagents were supplied in unit doses, thereby eliminating the need for precise pipetting. The extraction tube was then capped and used as a dropper to deliver 5 drops (∼100 μl) of the extracted sample to a tube containing the lyophilized amplification and detection reagents. The resulting mixture was agitated gently until a clear pink solution was obtained, after which the test strip, coated with a monoclonal antibody to chlamydial lipopolysaccharide and including a procedural control, was added to the solution and allowed to stand for 25 min before the result was read. Each swab was subjected to extraction twice in order to ensure that chlamydial antigen was extracted optimally from the viscous VS sample. Both CRT test strips were used in the interpretation of the result; a clearly visible test line on one or both test strips indicated a positive result, provided that the control lines were also visible on each test strip.

Treatment of PCR-positive participants.Participants who tested positive for C. trachomatis by PCR were recalled to the clinic and offered antibiotic treatment according to the guidelines of the Philippine Department of Health: 200 mg of ofloxacin and 100 mg of doxycycline twice daily for 14 days from 2002 through 2003 or a single dose (1 g) of azithromycin (from 2004 through 2006). Pregnant women positive for C. trachomatis by PCR were administered 500 mg amoxicillin three times a day for 7 days.

Statistical analysis.Data analysis was performed with SAS version 9.1 software. The 95% confidence intervals (CI) for sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were calculated as an exact binomial. The prevalence of C. trachomatis infection at each site was calculated on the basis of PCR positivity, with the 95% CI being calculated according to the Wald method, and differences were statistically compared by chi-square test. Differences in the proportion of positive test results in a subset of data from paired specimens in June 2004 were assessed with McNemar's test. The Mantel-Henszel trend test was used to analyze the NGU, gonorrhea, and syphilis prevalence rates recorded by the SHC.

SHC.Chlamydia testing by PCR was performed on a total of 2,015 (1,036 VS and 979 CS) specimens from FSWs (Table 1). Matched VS and CS specimens were obtained from participants attending the clinic in 2002 and in June 2004 as well as from FSWs in 18 different outreach settings in Iloilo City in November 2002. FSWs visit the SHC regularly for routine STI testing to comply with a government requirement for their profession. From 2000 to 2004, the SHC recorded annual prevalence rates of 10.5 to 26.0% for NGU, 1.5 to 3.7% for gonorrhea, and 2.1 to 5.4% for syphilis, with syphilis and gonorrhea showing significantly decreasing rates over time (P = 0.0079 and P < 0.0001, respectively). In contrast, NGU showed a significant increase in prevalence (P < 0.0001) over the same time period.

The study was primarily aimed at determining the prevalence of C. trachomatis infection by PCR in these two sites. On certain testing occasions, when either Clearview or the CRT was available, the performance and utility of these two rapid tests were also determined.

The combined data from all patients (n = 1,484) attending the SHC using both specimen types indicated that the prevalence of C. trachomatis infection ranged from 17.9% (35/196) to 20.7% (62/300) in 2002 and 2004, increased to 32.0% (93/291) in 2005, and then decreased to 22.0% (47/214) in 2006 (P = 0.0002 for 2002 to 2004 versus 2006; Table 1). Analysis of C. trachomatis positivity rate by sample type revealed no significant difference over the study period for CS specimens (P = 0.87), but a statistically significant increase for VS specimens was apparent in 2005 (P = 0.002). In a subset of specimens consisting of paired CCVSs and CSs collected in 2004, C. trachomatis positivity determined from CCVS specimens from FSWs did not differ significantly (P = 0.2482) from that for CS specimens (Table 2).

With PCR as the reference test, the sensitivity and specificity of the Clearview rapid test were 53.8% (85/158,) and 99.1% (657/663) for CS specimens, respectively, and 31.1% (19/61) and 95.2% (259/272) for VS specimens, respectively (Table 2). The PPV and NPV of the Clearview rapid test were 93.4 and 90.0%, respectively, for CS specimens and 59.4 and 86.0%, respectively, for VS specimens (Table 2). In comparison, CRT testing of the paired VS specimens showed a sensitivity of 71.0% (66/93) and a specificity of 99.0% (196/198), with a PPV and NPV of 97.1 and 87.9%, respectively (Table 3).

The SHC attendees during the study period were derived from at least 18 registered nightclubs and massage parlors that employed approximately 310 FSWs. Demographic analysis of a subset of these women (n = 782, from June 2004 to March 2006) revealed an age range of 18 to 56 years (median, 25.8 years). Most of the study participants were asymptomatic at the time of specimen collection, with only 28% reporting genitourinary symptoms. Self-reported methods of contraception included condom use by partner (53%); oral contraception (12%); other means such as tubal ligation, intrauterine device, or depot medroxyprogesterone acetate (1%); and none (34%).

A subset of participants who were positive for C. trachomatis infection by PCR in October 2005 and who had been treated were followed up and retested in March 2006. Of the 88 women who had originally tested positive, 16 were retested and 4 (25%) were again found to be positive for C. trachomatis by PCR.

OB-GYN clinic.Most of the participants at the OB-GYN clinic were pregnant women attending for routine antenatal care. The prevalence of C. trachomatis infection in this “low-risk” population was 6.3% (53/838) on the basis of PCR testing of VS specimens. Taking the PCR results as reference, the CRT showed a sensitivity and specificity of 86.8% (46/53) and 99.6% (782/785), respectively, with a PPV and NPV of 93.9 and 99.1%, respectively (Table 3).

The rates of PCR inhibition for the SHC and the OB-GYN samples were 2.4% (7/291) and 5% (42/838), respectively, during the 2005 and 2006 testing. Further processing by heating (at 95°C for 10 min) of inhibitory samples resulted in the resolution of 6 negatives and 1 positive for SHC and 41 negatives and 1 positive for OB-GYN. In the earlier testing dates at the SHC, when further processing by heating was not performed, about 4% of the inhibitory samples were unresolved and excluded from the study.